Mechanisms that Govern Cardiomyocyte Proliferation and Remuscularization following Ventricular Injury

摘要 心肌损伤后心肌细胞增殖和再肌化的调控机制 计划总体 心肌梗死治疗的总体目标是替换因缺血损伤造成的疤痕组织 有功能的心肌。由于成年哺乳动物心肌细胞(CMS)是非增殖的，并且 心脏细胞疗法的植入率极低，大多数肌肉化倡议如下 脑梗塞一直没有成功。然而，最近来自我们实验室的初步研究使用新生儿 猪已经证明，当心肌梗死(MI)在出生后第1天(P1)被诱导时，CMS重新进入细胞 循环，增殖，并完全恢复心脏功能，几乎没有疤痕。此外，我们发现， 这些患有P1损伤的新生儿心脏，具有非常活跃和持续的CM增殖机制，并且 因此，在P28处发生第二次LAD结扎损伤，导致LAD后第2-7天出现大面积梗塞(TTC 结扎，伤后4周未见明显脑梗塞。正如它所展示的那样，这是一个了不起的结果， 第一次，大型哺乳动物的心脏可以通过CM增殖使梗死的心脏组织重新肌化。 组成该计划项目赠款(PPG)申请的研究将检查CMS 重新进入细胞周期和使受伤的心脏重新肌化的新策略。项目1将确定CM细胞周期 由一天龄猪心肌梗死激活的调节剂，并构建人心肌斑块(HCMP) 来自具有激活细胞周期的增殖层的HiPSC-CMS前所未有的临床相关维度 随后的实验将确定已识别的因子和 心肌梗死后，hCMPS可使成年猪心脏再肌化。项目2将使用遗传策略、病毒载体、 并修改RNA以调查Sonic Hedgehog信号通路的成员是否包括Gli1 和Sox4，已被证明可以诱导培养的CMS增殖，并将促进CM的增殖 在成年小鼠和猪受伤的心脏中。此外，研究还将检查总监管者的能力， ETV2，以促进新生血管，促进成年小鼠和猪受损心脏的修复。项目3 将是先前的观察的延伸，即哺乳动物的细胞周期停滞至少部分是由 出生后发生的氧代谢增加，严重的全身缺氧会上调脯氨酸 在成年小鼠体内代谢并诱导CM增殖；拟议的研究将检查Pro 慢性低氧时代谢调节CM的存活和增殖。这三个项目加在一起， 相关的核心和调查人员的专业知识将加速和扩大研究，以解决 这一P01提案的中心目标：通过促进 内源性CM增殖，以及通过移植已准备好的功能性成熟的hCMP从“外部” 体内CM增殖。

ABSTRACT Mechanisms that govern cardiomyocyte proliferation and remuscularization following ventricular injury Program Overall The overall goal for the treatment of myocardial infarction is to replace the scar tissue caused by ischemic injury with functional cardiac muscle. Since adult mammalian cardiomyocytes (CMs) are non-proliferative, and the engraftment rate for cardiac cell therapy is extremely low, most of the remuscularizing initiatives following infarction have been unsuccessful. However, recent preliminary studies from our laboratories using neonatal pigs have shown that when myocardial infarction (MI) is induced on postnatal day 1 (P1), CMs re-enter the cell cycle, proliferate, and completely restore cardiac function with little scarring. Furthermore, we have found that these neonatal hearts with the P1 injury, have a very active and prolonged CM proliferative machinery, and consequently a second LAD ligation injury at P28, which resulted in a large infarct (TTC) at Day 2-7 post LAD ligation, produced no visible infarct 4 weeks following injury. This was a remarkable result as it demonstrated, for the first time, that a heart of large mammal could remuscularize infarcted heart tissue by CM proliferation. The studies comprising this Program Project Grant (PPG) application will examine mechanisms whereby CMs reenter the cell cycle and new strategies to remuscularize injured hearts. Project 1 will identify the CM cell-cycle regulators that are activated by MI in one-day-old pigs and construct human cardiac muscle patches (hCMP) of unprecedented clinically relevant dimensions from layers of proliferating hiPSC-CMs with activated cell cycle regulators, and other cardiac cells; subsequent experiments will determine whether the identified factors and hCMPs can remuscularize the hearts of adult pigs after MI. Project 2 will use genetic strategies, viral vectors, and modified RNAs to investigate whether members of the Sonic Hedgehog signaling pathway including Gli1 and Sox4, which have already been shown to induce proliferation in cultured CMs, will promote CM proliferation in the injured hearts of adult mice and pigs. In addition, studies will examine the capacity of the master regulator, Etv2, to promote neovascularization and promote repair of the injured hearts of adult mice and pigs. Project 3 will be an extension of previousobservations that mammalian cell-cycle arrest is at least partially induced by the increase in oxygen metabolismthat occurs after birth, and that severe systemic hypoxia upregulates proline metabolism and induces CM proliferation in adult mice; the proposed studies will examine whether proline metabolism regulates CM survivaland proliferation during chronic hypoxia. Collectively, these three projects, the associated cores and the expertise of the investigators will accelerate and amplify the studiesto address the central objective of this P01 proposal: To remuscularize the injured ventricle from “within,” by promoting endogenous CM proliferation, and from “outside,” bytransplanting functionally mature hCMPs that are primed for in-vivo CM proliferation.